Immunoreactivity of VR1 on epidermal keratinocyte of human skin.

We demonstrated the immunoreactivity of the receptor proteins, VR1, ion channels associated with pain sensation, on the epidermis of the human skin. Immunohistochemistry using antiserum against VR1 derived peptide showed immunoreactivity on the keratinocytes cell membrane of the human epidermis and cultured keratinocytes. The blocking peptide of the antiserum reduced the immunoreactivity on the epidermis. RT-PCR assay of cultured human keratinocyte also showed expression of VR1 mRNA. These results suggest the existence of VR1-like protein in epidermal keratinocytes of human skin.

Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney.

The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.

Functional characterization of structural alterations in the sequence of the vasodilatory peptide maxadilan yields a pituitary adenylate cyclase-activating peptide type 1 receptor-specific antagonist.

Maxadilan is a vasodilatory peptide derived from sand flies that is an agonist at the pituitary adenylate cyclase-activating peptide (PACAP) type 1 receptor. Surprisingly, maxadilan does not share significant sequence homology with PACAP. To examine the relationship between structure and activity of maxadilan, several amino acid substitutions and deletions were made in the peptide. These peptides were examined in vitro for binding to crude membranes derived from rabbit brain, a tissue that expresses PACAP type 1 receptors; and induction of cAMP was determined in PC12 cells, a line that expresses these receptors. The peptides were examined in vivo for their ability to induce erythema in rabbit skin. Substitution of the individual cysteines at positions 1 and 5 or deletion of this ring structure had little effect on activity. Substitution of either cysteine at position 14 or 51 eliminated activity. Deletion of the 19 amino acids between positions 24 and 42 resulted in a peptide with binding, but no functional activity. The capacity of this deletion mutant to interact with COS cells transfected with the PACAP type 1 receptor revealed that this peptide was a specific antagonist to the PACAP type 1 receptor.

Identification of osteopontin as a novel ligand for the integrin alpha8 beta1 and potential roles for this integrin-ligand interaction in kidney morphogenesis.

Epithelio-mesenchymal interactions during kidney organogenesis are disrupted in integrin alpha8 beta1-deficient mice. However, the known ligands for integrin alpha8 beta1-fibronectin, vitronectin, and tenascin-C-are not appropriately localized to mediate all alpha8 beta1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for alpha8 beta1. We have coexpressed the extracellular domains of the mouse alpha8 and beta1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the alpha8 beta1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with alpha8 beta1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In "far Western" blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to alpha8 beta1-AP. Cell adhesion assays using K562 cells expressing alpha8 beta1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin alpha8 beta1 may help regulate kidney development and other morphogenetic processes.

[Anesthetic management for renal tumor extending to the inferior vena cava].

A 58 year-old woman underwent radical nephrectomy, thrombectomy and ileo-cecal resection for renal tumor with thrombus involving the inferior vena cava and ascending colon cancer. In a patient having tumor thrombus extending to the vena cava, recognition of the position of the thrombus is important for surgical and anesthetic management in pre- and intra-operative periods. Transesophageal echocardiography (TEE) enabled us to visualize the real-time movement and deformity of thrombus by surgical manipulation and compression during operation. TEE seemed also very useful not only in understanding the hemodynamics during operation but also in detecting the residual tumor and the blood flow in liver and the inferior vena cava after operation.

Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C.

The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.

[Anesthetic managements for pericardiectomy of three patients with constrictive pericarditis].

We experienced three patients with constrictive pericarditis who underwent pericardiectomy. Percutaneous cardiopulmonary support (PCPS) was carried out in two patients because of unstable hemodynamics caused by massive bleeding or cardiac compression due to surgical manipulation. In the other patient with severe tachycardia, we prepared PCPS before the induction of anesthesia, and could manage the whole course of anesthesia satisfactorily. It is suggested that PCPS is the most reliable way to support hemodynamics during anesthesia in patients with constrictive pericarditis.

Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis.

We present genetic evidence that integrins regulate epithelial-mesenchymal interactions during organogenesis. Mice with a mutation in the alpha8 gene do not express the integrin alpha8 beta1 and exhibit profound deficits in kidney morphogenesis. In wild-type animals, inductive interactions between the ureteric epithelium and metanephric mesenchyme are essential for kidney morphogenesis. In alpha8 mutant homozygotes, growth and branching of the ureteric bud and recruitment of mesenchymal cells into epithelial structures are defective. Consistent with these phenotypes, alpha8 expression is induced in mesenchymal cells upon contact with the ureter. Since none of its previously identified ligands appears likely to mediate the essential functions of alpha8 beta1 in kidney morphogenesis, we have used an alpha8 beta1-alkaline phosphatase chimera to localize novel ligand(s) in the growing ureter. The distribution of these ligand(s) makes them strong candidates for regulators of kidney morphogenesis.

Somatosensory evoked potentials recorded from the posterior pharynx to stimulation of the median nerve and cauda equina.

Somatosensory evoked potentials (ppSEPs) in response to stimulation of the median nerve at the wrist and the cauda equina at the epidural space (the L4 level) were recorded from the posterior wall of the pharynx in 15 patients who underwent spinal surgery under general anesthesia, using disc electrodes attached to the endotracheal tube, and compared with segmental spinal cord potentials (seg-SCPs) that were recorded simultaneously from the posterior epidural space (PES). ppSEPs consisted of the initially positive spike (P9) followed by slow positive (P13) and negative (N22) waves. The P13 and N22 of ppSEPs had phase reversal relationship with the P2 and N2 recorded from the PES, respectively. The peak latencies of P9 (9.40 +/- 0.7 ms) (mean +/- SD), P13 (13.1 +/- 0.9 ms), and N22 (22.0 +/- 2.1 ms) of ppSEPs coincided with those of P1, N1 and P2 of seg-SCPs, respectively, ppSEPs were recorded more clearly with a reference electrode on the dorsal surface of the neck than with the reference electrode at the earlobe or back of the hand. The threshold and maximal stimulus intensities were also similar between the ppSEPs and seg-SCPs. Thus, the P9, P13, and N22 components of ppSEPs were thought to have the same origin as the P1, N1 and P2 of seg-SCPs, respectively. Therefore, the P9, P13 and N22 of ppSEPs may reflect incoming volleys through the root, synchronized activities of the interneurons and primary afferent depolarizations (PAD), respectively. ppSEPs in response to cauda equina stimulation showed that the latencies of the two initial components (4.6 +/- 0.4 and 6.4 +/- 0.6 ms) corresponded to those of the SCPs recorded from the PES (4.6 +/- 0.3 and 6.3 +/- 0.5 ms), suggesting that these potentials reflect impulses conducting through the spinal cord, similar to epidurally recorded SCPs.

[Paraplegia following coeliac plexus block by anterior approach under direct vision].

We report here a case of paraplegia following coeliac plexus block by anterior approach under direct vision. Laparotomy was performed in a 62-year-old male patient with pancreatic cancer. Coeliac plexus block was undertaken in order to control his back pain, since the tumor was unresectable. The patient complained of numbness and weakness of his legs 14 hours later. The consequence of neurological events was diagnosed as ischemic infarct of the spinal cord by myelo-CT and MRI. He died of pancreatic cancer without recovery of neurological disturbances 4 months after the surgery. As demonstrated in this case, even when coeliac plexus block was performed by open anterior approach under direct vision, paraplegia might be a possible complication due to the anatomical proximity of coeliac plexus to the Adamkiewicz's artery.

Central nuclei and spinal pathways in feedback inhibitory spinal cord potentials in ketamine-anaesthetized rats.

It has been suggested that heterosegmentally activated slow positive potentials (HSP), recorded from the spinal cord of rat and humans, are feedback inhibitory potentials. The present study was carried out to define ascending and descending spinal tracts and the sites of central nuclei involved in the production of these HSP, and the effects of ketamine on these central nuclei. The spinal cords in ketamine-anaesthetized rats were transected to determine the ascending and descending tracts involved in the production of hindpaw (HP) and forepaw (FP) HSP, respectively. Lesions of the brain at various levels were performed stereotactically during ketamine anaesthesia. Dorsal one-third resection of the cord at the T8-9 level did not affect HSP significantly, while contralateral lesion of the dorsal two-thirds of the cord decreased FP-HSP but not HP-HSP during ketamine. Bilateral transection of the ventral one-third of the cord abolished both HSP. Ablation of the cerebral cortex, cerebellum, thalamus, midbrain and pons did not affect HSP significantly. However, transection of the middle medulla decreased, while transection of the most caudal part of the medulla completely abolished both HSP. Ketamine decreased HSP even in the medulla-spinal cord preparation and the segmental slow positive wave in spinalized animals. In ketamine-anaesthetized rats, ascending and descending spinal tracts involved in the production of HP-HSP and FP-HSP are located bilaterally in the ventrolateral quadrant and in the contralateral lateral funiculus and ventrolateral quadrant, respectively. Principal central nuclei feeding back HSP might be situated diffusely in the medulla down to the caudal part. Ketamine is suggested to suppress these inhibitory feedback potentials predominantly at, and partly even below, the level of the medulla.

Structural characterization and location of disulphide linkages of a potent vasodilatory peptide, recombinant maxadilan, by a multiple mass spectrometric approach.

A multiple mass spectrometric strategy using fast-atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) has been used to confirm the sequence and to locate the disulfide linkages of recombinant maxadilan (r-maxadilan) (average molecular mass 7422.5 Da), a potent vasodilatory peptide from Lutzomyia longipalpis. MALDI measurements of intact r-maxadilan, its reduced form and its pyridylethylated form (p-maxadilan) indicated the presence of four Cys residues without major post-translational modifications. FAB and FAB-tandem mass spectrometry measurements of chymotryptic digests of p-maxadilan were sufficient to map the primary structure of p-maxadilan, though the complementary use of MALDI was necessary for complete mapping using Asp-N digestion due to a strong suppression observed in FAB. Assignment of the Cys-5-Cys-9 linkage was achieved by comparison of FAB mass spectra before and after reduction of tryptic digests of r-maxadilan. Since the molecular weight of the peptide fragment containing the Cys-18-Cys 55 linkage is more than 4000, MALDI measurement was indispensable for assignment of this linkage. The results fully support the value of the multiple mass spectrometric strategy in the structural characterization of peptides and proteins.

Spinal tracts producing slow components of spinal cord potentials evoked by descending volleys in man.

Slow negative (N) and slow positive (P) waves are frequently produced in the posterior epidural space at the lumbosacral enlargement by epidural stimulation of the rostral part of human spinal cord. The production of these slow potentials are thought to be responsible for analgesia at the stimulated segment as well as below that level. In order to define the spinal tract which mediates these slow potentials, we stimulated directly or from the epidural space the dorsal, dorsolateral, lateral and ventral columns at the cervical or thoracic level, and epidurally recorded spinal cord potentials (des.SCPs) at the lumbosacral enlargement in 7 patients who underwent spine or spinal cord surgery. The des.SCPs recorded in the lumbosacral enlargement consisted of polyphasic spike potentials followed by slow N and P waves. At a near threshold level of stimulus intensity the slow N and P potentials were consistently elicited only by stimulation of the dorsal column. The slow waves were also produced by intense stimulation of other tracts, but remained significantly (P < 0.05 - P < 0.01) smaller than those evoked by dorsal column stimulation when compared at the same stimulus intensity. Moreover, the slow P wave could not be elicited even by intense stimulation (10 times the threshold strength for the initial spike potentials) of the ventral column. Thus, the results suggest that the slow N and P waves are mostly mediated by the antidromic impulses descending through the dorsal column.

Spinal cord potential recordings from the extradural space during scoliosis surgery.

For monitoring spinal cord functions during corrective surgery of scoliosis, we have recorded percutaneously from the posterior extradural space at the C5-7 levels the ascending conducted spinal cord potentials (ASCP) in response to extradural stimulation of the cauda equina in 134 patients. The ASCP consists of three spike-like components (C1, C2 and C3) followed by slow components. The extradurally recorded ASCP were not affected by anaesthetic agents. There were no significant differential effects of spinal distractions on each of the three spike potentials. There were no post-operative neurological abnormalities in patients whose ASCP showed no changes, amplitude increases, amplitude decreases of less than 50% or latency increases (> 0.2 ms) during spinal manipulations (no false negatives, but some false positives). Five patients who suffered postoperative neurological damage exhibited more than 50% changes in amplitude of the ASCP during surgery. All these neurological sequelae occurred in the first 80 patients. In the last 54 patients, in whom the distraction forces on the spine were controlled rapidly by observation of the amplitude changes in ASCP, there were no postoperative neurological abnormalities, except for one patient in whom an accidental spinal cord injury was produced by a hook. The results suggest that the distraction force on the spine must be reduced immediately when the amplitudes of the ASCP decrease by more than 50% of control values with or without latency increases.

Effects of pentobarbital on heterosegmentally activated dorsal root depolarization in the rat. Investigation by sucrose-gap technique in vivo.

Slow positive cord dorsum (P-) potentials activated by segmental stimulation are believed to reflect primary afferent depolarizations and have been shown to be augmented by barbiturates. However, there have been no data to confirm whether heterosegmentally activated P-potentials also represent primary afferent depolarizations and are similarly affected by barbiturates. We therefore tested whether heterosegmental P-potentials reflect primary afferent depolarizations and how these heterosegmental potentials are affected by barbiturates. Heterosegmentally activated dorsal root (DR) depolarizations (depolarizations evoked in DRs of lumbar segments in response to afferent volleys to cervical segments produced by electrical stimulation of the forepaw) and P-potentials were simultaneously recorded, adapting the sucrose-gap technique for recording DR depolarization in vivo in the rat. Forepaw (heterosegmental) stimulations produced a large depolarization in the DRs of L5-S1 as well as a slow P-potential in the lumbosacral enlargement. Transection of the spinal cord at the level of C1-C2 abolished both the P-potential and DR depolarization activated by heterosegmental stimulation as well as the second component of segmentally (hind-paw) activated P-potential. Bicuculline (100 micrograms/kg, intravenous) augmented the P-potential and DR depolarization produced by heterosegmental stimulation, but larger doses, 400-600 micrograms/kg, eventually suppressed these. However, the drug, in a dose-dependent manner, suppressed both the P-potential and DR depolarization produced by the segmental stimulation. Pentobarbital (10-40 mg/kg, intravenous) suppressed in a dose-dependent manner both the heterosegmental P-potential and heterosegmental DR depolarization and prolonged their peak latencies. By contrast, pentobarbital augmented and prolonged the segmental P-potential and segmental DR depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Slow positive dorsal cord potentials activated by heterosegmental stimuli.

Heterosegmental slow positive waves (HSPs) and segmental spinal cord potentials were recorded from the cord dorsum in ketamine-anesthetized rats. Forepaw stimulation produced HSPs in the lumbo-sacral enlargement (lumbar HSPs), whereas hind paw stimulation evoked HSPs in the cervical cord (cervical HSP). Both the HSP and the secondary component of the slow positive wave (P2s) in the segmental spinal cord potential were highly vulnerable to anesthetics and completely disappeared after spinal cord transection at the C1/2 level, indicating that both the HSP and P2s are produced by a long feedback loop via supraspinal structures. The lumbar HSP evoked by forepaw stimulation was maximal in amplitude at the L5 level and more dominant in the ipsilateral cord dorsum than in the contralateral one, but widely distributed in the lumbo-sacral cord. A variability of onset (7-18 msec for cervical and 5-17 msec for lumbar HSPs) and peak (22-35 msec for cervical and 12-50 msec for lumbar HSPs) suggests the existence of several nuclei to form the feedback loops for descending impulses to produce the HSPs. There were no peak latency differences between the HSPs and P2s. Since there were several similar characteristics between the P2s and HSP such as a high vulnerability to anesthetic, a complete disappearance after high spinal transection and similar response curves to graded intensities of stimulation, there may be a close relationship between their feedback nuclei and the pathways mediating them. All wide dynamic range (WDR) neurons (12/12) in lamina V of Rexed responded to heterosegmental stimulation with inhibition of firing.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dorsal root entry zone lesion on spinal cord potentials evoked by segmental, ascending and descending volleys.

The spinal cord potentials (SCPs) were recorded from the dorsal root entry zone (DREZ) and posterior epidural space in patients before and after dorsal root entry zone lesion (DREZL) during general anaesthesia. The SCPs from the DREZ activated by segmental, ascending and descending volleys were basically the same in fundamental waveform as those recorded from the posterior epidural space. Segmentally activated slow negative (N1) wave, reflecting synchronized activities of dorsal horn neurones, and positive (P2) wave, thought to indicate primary afferent depolarization, were affected by DREZL in all 4 subjects tested, even by contralateral stimulation, suggesting that these components of the segmental SCPs in man partly reflect the activities of the contralateral dorsal horn. The spike-like potentials activated by ascending volleys were not affected by DREZL, while the subsequent slow components were decreased in the lesioned level. This may indicate that ascending spinal cord tracts are not affected by the operation, and suggests that the origin of the slow components by ascending volleys lies at least in part in the segmental dorsal horn. The slow negative and positive components, recorded at a remote segment from DREZL, in response to the descending volleys, were augmented after DREZL, suggesting that activation of ascending or descending inhibition through a feedback loop via the supraspinal structures might occur at least transiently following DREZL. All components of the SCPs activated by descending volleys were decreased or disappeared in recording from the lesioned level, as expected. Thus, intra-operative recording of the SCPs during DREZL might be beneficial for monitoring and studying human spinal cord function.

[Anesthetic management of pheochromocytoma by continuous intravenous injection of prostaglandin E1].

We had five cases of surgical removal of pheochromocytoma by continuous intravenous injection of prostaglandin E1. During anesthesia, we used Swan-Ganz catheter for circulatory monitoring and measured plasma catecholamines. When PGE1 dose was increased from 0.05 to 0.1 and 0.15 microgram.kg-1.min-1, total systemic vascular resistance and mean arterial pressure were decreased but heart rate and cardiac output were not significantly altered from the preanesthetic values. Plasma catecholamines were also similar to the preanesthetic values. Therefore, the results suggest that the mechanism of suppression of hypertension by PGE1 is by affecting vascular beds directly rather than by diminishing catecholamine excretion from sympathetic nerve and adrenal medulla. During manipulation of pheochromocytoma, mean arterial blood pressure increased extremely. Although PGE1 was injected at a rate of 0.3 to 0.5 microgram.kg-1.min-1 in some cases, we could not suppress the elevation of blood pressure. PGE1 alone could not normalize blood pressure and heart rate, and other cardiovascular agents were necessary.